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Mo1676 Flagellar Glycosylation Influences Motility, Flagellar Assembly, and Toxin Production in Clostridium difficile
most effective when added at 10 nmM concentration and also if added to the Caco-2 cells immediately after reaching confluency. Improved barrier function induced by Vitamin D led to a decrease in cell rounding/apoptosis in the presence of low concentrations of C. difficle toxin A, while little protective effect was observed at higher concentrations of toxin A. It was noted that the addition of vitamin D resulted in a morphologically distinct Caco2 cellular membrane bleb with numerous epithelial projections, and studies to identify the mechanism for Vitamin A-induced phenotypic changes are underway. Conclusion Our experiments indicates that addition of vitamin D to Caco-2 cells leads to improved cell barrier function and it is also protective against low concentrations of C. difficile toxin A. The underlying increased TEER and protective effect against C. difficile toxin A maybe due to increased tight junctions and cellular structural transformation seen under microscopy in Caco-2 cells in presence of vitamin D.
Mo1675 A Caenorhabditis elegans Model to Study Mechanisms of Microbiota Mediated Protection Against Clostridium difficile Teklu K. Gerbaba, Amtul Moeez, Ian D. Chin Sang, Elaine O. Petrof Background: Clostridium difficile, a toxin producing bacterium, is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis. By destroying the diversity of the microbiota, antibiotic treatment creates an environment conducive for C. difficile proliferation and toxin production, rendering the host susceptible to C. difficile infection. Although the importance of a healthy microbiota for defense against C. difficile infection is well established, the key protective species (and mechanisms of protection) remain poorly understood. Aims: To develop a simple and high-throughput applicable model for the study of mechanisms of microbiota-mediated protection and for the discovery of novel microbial therapeutics against C. difficile toxins. Methods: Age synchronized L1 worms were grown to L4 stage by feeding E. coli OP50 in the presence or absence of antibiotics (Carbenicillin). L4 stage worms were treated with C. difficile toxin (TcdB) in a 96 well plate and worm survival was monitored. The effect of toxin treatment on cytoskeletal structure of the intestine was determined by FITC-phalloidin staining. Toxin effect on gut barrier/integrity was determined by FITC-Dextran assay. Results: Pre-exposure of worms to E. coli OP50 with antibiotics renders worms susceptible to C. difficile toxin, whereas antibiotic non-exposed worms remained resistant (n = 90; log rank test, p < 0.05). FITC-phalloidin staining of toxin treated worms revealed decreased apical actin, destabilization of intestinal cytoskeletal structures and distension of the gut lumen. Toxin treatment also resulted in intestinal barrier breach as demonstrated by FITC-Dextran assay. FITC-Dextran was restricted to the intestinal lumen and was not seen in the pseudocoelom of controls, whereas toxin treated worms showed leakage of FITC-Dextran into the pseudocoelom. Co-treatment of worms with toxin in the presence of secreted/excreted products of a consortium of 33 healthy human gut microbial isolates protected worms against the effect of toxins on apical actin, intestinal barrier function and survival in antibiotic pretreated worms. Conclusions: The developed model system would be useful to study mechanisms of microbiota-mediated protection and to discover novel microbial therapeutics against C. difficile toxins.
Mo1673 Discovery of a Toxin-Positive Non-Pathogenic Clostridium difficile Isolate That Protects Against Pathogenic C. difficile Lucie Etienne-Mesmin, Benoit Chassaing, Oluwaseyi Adekunle, Adrianne N. Edwards, Shonna M. McBride, Andrew T. Gewirtz BACKGROUND: Clostridium difficile infection is the leading cause of nosocomial infections and antibiotic-associated diarrhea in United States. This spore-forming obligate anaerobic bacterium is a difficult pathogen to treat and to remove from the environment as it forms stable spores. C. difficile infection in mice is modeled by administration of antibiotic cocktails followed by exposure to C. difficile spores. Previous studies conducted in our lab and by others have observed a variability of infectivity depending on the vivaria. GOAL The aim of the current study was to understand such infectivity variability by analyzing the gut microbiota of mice susceptible versus resistant to C. difficile infection. METHODS: Pathogenesis of C. difficile strains (well-characterized pathogenic C. difficile strain VPI 10463 and an ubiquitous murine C. difficile isolate) were investigated using antibiotic-treated mice (oral kanamycin, metronidazole, gentamicin, vancomycin, colistin; intra-peritoneal injection of clindamycin) and germ free mice were used for mono/bi-association studies. C. difficile genomic copy number in feces was quantified by q-PCR and C. difficile-associated toxins secretion were analyzed using Vero-cells assay. Bacterial strain sequencing was performed on purified DNA using a PacBio RS II apparatus. RESULTS: Following antibiotic treatment, a substantial percentage of mice purchased from major suppliers exhibited a bloom of an endogenous C. difficile strain whose presence correlated with levels of C. difficile toxin but not any indicators of disease. Importantly, such blooms correlated with mice being protected against virulent C. difficile strain VPI 10463 infection. The ubiquitous but furtive C . difficile harbored in the murine intestine was isolated on taurocholate-cefoxitin-cycloserine-fructose agar medium, and demonstrated to sporulate and produce toxin while ribotyping indicated it is a previously uncharacterized C. difficile strain, we have named LEM1. While infection of antibiotic-treated mice with the pathogenic C. difficile strain VPI 10463 results in weight loss and lethargy progressing to death, mice pre-colonized with the C. difficile murine isolate LEM1 are fully protected from disease. Moreover, C. difficile VPI 10463 causes an acute and lethal form of infection in germfree mice, while mice mono-associated with C. difficile strain LEM1 remained fully healthy even when administered high doses of C. difficile VPI 10463 spores. CONCLUSIONS: We have isolated a very minor but common component of the mouse microbiota, namely an avirulent strain of C. difficile that lacks pathogenicity and, moreover, protects against highly virulent C. difficile. Full genome sequencing of the protective strain was performed, and its analysis will help to mechanistically decipher the protective effect.
Mo1676 Flagellar Glycosylation Influences Motility, Flagellar Assembly, and Toxin Production in Clostridium difficile Gayatri Vedantam, Aaron Brussels, Andrew Clark, V.K. Viswanathan Clostridium difficile (CD) has emerged as the most prevalent healthcare-associated infection, responsible for an estimated annual cost of $3 billion to the US healthcare system. Disease is mediated through the action of two glucosylating toxins which are relatively well-studied; however, even basic host colonization mechanisms remain largely undefined. The CD genome encodes a number of characterized and putative colonization factors including a type IV pilus, fibronectin binding proteins, surface layer proteins, and a flagellar system. Goal: CD flagella are surface structures critical for bacteria-host interactions. The aim of this work was to assess the impact of post-translational modification on flagellar organelle assembly and function. Methods and Results: CD flagellar structures were visualized and shown to be glycosylated via specific glycoprotein-staining electron microscopy. Analyses of publiclyavailable CD genomes revealed two clade-specific glycosylation islands within the CD flagellar biosynthesis locus predicted to modify the structural protein flagellin (FliC). Disruption mutagenesis as well as gene-swapping studies of glycosylation genes within these islands showed that FliC modification was necessary for flagellar assembly and motility, and that it impacted CD toxin secretion as well as bacterial adherence. Electron transfer disassociation mass spectrometry (ETD-MS) revealed specific glycan moieties in CD strain BI-1 (ribotype 027), enabling the construction of a model for FliC glycosylation in this clade background. Conclusions: CD flagellin post-translational modification is critical for appropriate organelle assembly and function, and likely, therefore, for pathogenesis. We have successfully generated multiple tools in this system which will enable further studies of disease establishment including, but not limited to, assessments of virulence in the hamster model of acute CDI.
Mo1674 Increased Transepithelial Electrical Resistance (TEER) of Caco-2 Cells in Presence of Vitamin D: Role of Intestinal Permeability in Clostridium difficile Infection Amir Kalani, Ashley Curatola, Michele Kutzler Introduction: Recent studies suggest a possible association between Vitamin D deficiency and increased Clostridium difficile infection (CDI). The underlying mechanism in which vitamin D levels directly affect Clostridium difficile pathogenesis has not been fully explored. Our primary aim was to characterize the effects of vitamin D administration on the permeability and viability of intestinal cells in vitro and to test our hypothesis that vitamin D exhibits a protective effect on cellular permeability and tight junction protein expression during C. difficile toxin-induced cellular apoptosis. Methods We used Caco-2 cells which morphologically and functionally resemble human enterocytes. Cells were grown on transwells until they reached confluency and polarization as measured by Transepithelial Electrical Resistance (TEER) and then were incubated with media alone or with media containing 1nM, 10 nM, or 100 nM 1a, 25-dihydroxyvitamin D3 at various points in cell growth cycle. Cell permeability was measured using TEER, the lucifer yellow diffusion assay and microscopy. After determining the optimal physiological concentration and incubation period for vitamin D administration, Caco-2 cells were incubated in absence and presence of various concentrations of C. difficile toxin A. Cellular tight junctions were directly visualized under fluorescent deconvolution microscopy after staining with tagged monoclonal antibodies against the tight junction proteins ZO-1 and F-actin. Cell lysates and supernatants were also collected for inflammatory cytokine analysis. Results Addition of vitamin D lead to decreased membrane permeability as measured by decreased lucifer yellow diffusion, increased TEER values and positive ZO-1/F-actin staining post treatment, indicating improved barrier function. It was
Mo1682 Characteristics of DNA Mismatch Repair Status and Tumor Distribution Patterns in Early-Onset Colorectal Cancer Patients Juliana F. Yang, Amy Noffsinger, Jayaprakash Sreenarasimhaiah, Qinghua Yang Background/Aim: Early-onset colorectal cancers (CRCs) often occur in patients with underlying colonic disease or hereditary/genetic abnormalities such as inflammatory bowel disease (IBD) or familial CRC (Lynch syndrome, LS). Immunohistochemical detection of DNA mismatch repair (MMR) proteins is a preliminary screening tool for LS, and may play a role in determining patient prognosis and treatment. Little is known about the relationship of MMR status and CRC anatomic locations in this group of patients. Methods: The Miraca Life Sciences (MLS) pathology database was queried for all primary CRCs from 1/1/2012 to 12/31/2014. Early-onset CRC patients were those £49 years of age. The control group
S-749
AGA Abstracts
AGA Abstracts
differences in other demographics. At the time of diagnosis men had higher frequencies of cirrhosis (8.7%, 4.0%, p<0.01), previous solid organ transplant (9.0%, 4.9%, p<0.05), PEG in place (10.9%, 6.6%, p<0.05), nasogastric or orogastric tube in place (29.4%, 17.8%, p<0.001), endotracheal intubation (16.5%, 8.4%, p<0.01), and active tube feeds (21.4%, 13.1%, p<0.01) compared with woman. Men received penicillin based antibiotics (49.2%, 40.1%, p<0.05) more frequently in the three months prior to diagnosis than women with no other differences in other medication exposures. Woman had a higher WBC at diagnosis (11.6±7.7, 12.9±8.7, p<0.05) but a lower Cr (1.7±1.7, 1.5±1.6, p<0.05). There were no significant differences in albumin at diagnosis. Men received longer durations of oral metronidazole than women (11.5±9.1, 10.1±6.0 days, p<0.01) but woman had a higher frequency of switching from vancomycin to metronidazole (0.0%, 1.5%, p<0.05). Woman were discharged to home (37.6%, 39.8%, p<0.01) and readmitted within 30-days with diarrhea (18.6%, 28.7%, p<0.05) more frequently than men. There were no other significant differences in short-term outcomes, including recurrence within 90-days. Men showed a trend toward a higher 30-day mortality rate (13.4%, 11.0%, p=0.29) compared with women. Men had a higher mortality rate after one year (34.3%, 25.8%, p<0.01). Conclusions: Men get CDI less frequently, but those who do get CDI, have higher co-morbidity. Treatment patterns are similar but men have a trend toward increased short-term mortality and a higher mortality rate at one year. This might be attributed to the increased medical co-morbidities of these patients and in men, CDI should be considered an indicator of worsened potential longterm outcomes.
Mo1675 A Caenorhabditis elegans Model to Study Mechanisms of Microbiota Mediated Protection Against Clostridium difficile Teklu K. Gerbaba, Amtul Moeez, Ian D. Chin Sang, Elaine O. Petrof Background: Clostridium difficile, a toxin producing bacterium, is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis. By destroying the diversity of the microbiota, antibiotic treatment creates an environment conducive for C. difficile proliferation and toxin production, rendering the host susceptible to C. difficile infection. Although the importance of a healthy microbiota for defense against C. difficile infection is well established, the key protective species (and mechanisms of protection) remain poorly understood. Aims: To develop a simple and high-throughput applicable model for the study of mechanisms of microbiota-mediated protection and for the discovery of novel microbial therapeutics against C. difficile toxins. Methods: Age synchronized L1 worms were grown to L4 stage by feeding E. coli OP50 in the presence or absence of antibiotics (Carbenicillin). L4 stage worms were treated with C. difficile toxin (TcdB) in a 96 well plate and worm survival was monitored. The effect of toxin treatment on cytoskeletal structure of the intestine was determined by FITC-phalloidin staining. Toxin effect on gut barrier/integrity was determined by FITC-Dextran assay. Results: Pre-exposure of worms to E. coli OP50 with antibiotics renders worms susceptible to C. difficile toxin, whereas antibiotic non-exposed worms remained resistant (n = 90; log rank test, p < 0.05). FITC-phalloidin staining of toxin treated worms revealed decreased apical actin, destabilization of intestinal cytoskeletal structures and distension of the gut lumen. Toxin treatment also resulted in intestinal barrier breach as demonstrated by FITC-Dextran assay. FITC-Dextran was restricted to the intestinal lumen and was not seen in the pseudocoelom of controls, whereas toxin treated worms showed leakage of FITC-Dextran into the pseudocoelom. Co-treatment of worms with toxin in the presence of secreted/excreted products of a consortium of 33 healthy human gut microbial isolates protected worms against the effect of toxins on apical actin, intestinal barrier function and survival in antibiotic pretreated worms. Conclusions: The developed model system would be useful to study mechanisms of microbiota-mediated protection and to discover novel microbial therapeutics against C. difficile toxins.
Mo1673 Discovery of a Toxin-Positive Non-Pathogenic Clostridium difficile Isolate That Protects Against Pathogenic C. difficile Lucie Etienne-Mesmin, Benoit Chassaing, Oluwaseyi Adekunle, Adrianne N. Edwards, Shonna M. McBride, Andrew T. Gewirtz BACKGROUND: Clostridium difficile infection is the leading cause of nosocomial infections and antibiotic-associated diarrhea in United States. This spore-forming obligate anaerobic bacterium is a difficult pathogen to treat and to remove from the environment as it forms stable spores. C. difficile infection in mice is modeled by administration of antibiotic cocktails followed by exposure to C. difficile spores. Previous studies conducted in our lab and by others have observed a variability of infectivity depending on the vivaria. GOAL The aim of the current study was to understand such infectivity variability by analyzing the gut microbiota of mice susceptible versus resistant to C. difficile infection. METHODS: Pathogenesis of C. difficile strains (well-characterized pathogenic C. difficile strain VPI 10463 and an ubiquitous murine C. difficile isolate) were investigated using antibiotic-treated mice (oral kanamycin, metronidazole, gentamicin, vancomycin, colistin; intra-peritoneal injection of clindamycin) and germ free mice were used for mono/bi-association studies. C. difficile genomic copy number in feces was quantified by q-PCR and C. difficile-associated toxins secretion were analyzed using Vero-cells assay. Bacterial strain sequencing was performed on purified DNA using a PacBio RS II apparatus. RESULTS: Following antibiotic treatment, a substantial percentage of mice purchased from major suppliers exhibited a bloom of an endogenous C. difficile strain whose presence correlated with levels of C. difficile toxin but not any indicators of disease. Importantly, such blooms correlated with mice being protected against virulent C. difficile strain VPI 10463 infection. The ubiquitous but furtive C . difficile harbored in the murine intestine was isolated on taurocholate-cefoxitin-cycloserine-fructose agar medium, and demonstrated to sporulate and produce toxin while ribotyping indicated it is a previously uncharacterized C. difficile strain, we have named LEM1. While infection of antibiotic-treated mice with the pathogenic C. difficile strain VPI 10463 results in weight loss and lethargy progressing to death, mice pre-colonized with the C. difficile murine isolate LEM1 are fully protected from disease. Moreover, C. difficile VPI 10463 causes an acute and lethal form of infection in germfree mice, while mice mono-associated with C. difficile strain LEM1 remained fully healthy even when administered high doses of C. difficile VPI 10463 spores. CONCLUSIONS: We have isolated a very minor but common component of the mouse microbiota, namely an avirulent strain of C. difficile that lacks pathogenicity and, moreover, protects against highly virulent C. difficile. Full genome sequencing of the protective strain was performed, and its analysis will help to mechanistically decipher the protective effect.
Mo1676 Flagellar Glycosylation Influences Motility, Flagellar Assembly, and Toxin Production in Clostridium difficile Gayatri Vedantam, Aaron Brussels, Andrew Clark, V.K. Viswanathan Clostridium difficile (CD) has emerged as the most prevalent healthcare-associated infection, responsible for an estimated annual cost of $3 billion to the US healthcare system. Disease is mediated through the action of two glucosylating toxins which are relatively well-studied; however, even basic host colonization mechanisms remain largely undefined. The CD genome encodes a number of characterized and putative colonization factors including a type IV pilus, fibronectin binding proteins, surface layer proteins, and a flagellar system. Goal: CD flagella are surface structures critical for bacteria-host interactions. The aim of this work was to assess the impact of post-translational modification on flagellar organelle assembly and function. Methods and Results: CD flagellar structures were visualized and shown to be glycosylated via specific glycoprotein-staining electron microscopy. Analyses of publiclyavailable CD genomes revealed two clade-specific glycosylation islands within the CD flagellar biosynthesis locus predicted to modify the structural protein flagellin (FliC). Disruption mutagenesis as well as gene-swapping studies of glycosylation genes within these islands showed that FliC modification was necessary for flagellar assembly and motility, and that it impacted CD toxin secretion as well as bacterial adherence. Electron transfer disassociation mass spectrometry (ETD-MS) revealed specific glycan moieties in CD strain BI-1 (ribotype 027), enabling the construction of a model for FliC glycosylation in this clade background. Conclusions: CD flagellin post-translational modification is critical for appropriate organelle assembly and function, and likely, therefore, for pathogenesis. We have successfully generated multiple tools in this system which will enable further studies of disease establishment including, but not limited to, assessments of virulence in the hamster model of acute CDI.
Mo1674 Increased Transepithelial Electrical Resistance (TEER) of Caco-2 Cells in Presence of Vitamin D: Role of Intestinal Permeability in Clostridium difficile Infection Amir Kalani, Ashley Curatola, Michele Kutzler Introduction: Recent studies suggest a possible association between Vitamin D deficiency and increased Clostridium difficile infection (CDI). The underlying mechanism in which vitamin D levels directly affect Clostridium difficile pathogenesis has not been fully explored. Our primary aim was to characterize the effects of vitamin D administration on the permeability and viability of intestinal cells in vitro and to test our hypothesis that vitamin D exhibits a protective effect on cellular permeability and tight junction protein expression during C. difficile toxin-induced cellular apoptosis. Methods We used Caco-2 cells which morphologically and functionally resemble human enterocytes. Cells were grown on transwells until they reached confluency and polarization as measured by Transepithelial Electrical Resistance (TEER) and then were incubated with media alone or with media containing 1nM, 10 nM, or 100 nM 1a, 25-dihydroxyvitamin D3 at various points in cell growth cycle. Cell permeability was measured using TEER, the lucifer yellow diffusion assay and microscopy. After determining the optimal physiological concentration and incubation period for vitamin D administration, Caco-2 cells were incubated in absence and presence of various concentrations of C. difficile toxin A. Cellular tight junctions were directly visualized under fluorescent deconvolution microscopy after staining with tagged monoclonal antibodies against the tight junction proteins ZO-1 and F-actin. Cell lysates and supernatants were also collected for inflammatory cytokine analysis. Results Addition of vitamin D lead to decreased membrane permeability as measured by decreased lucifer yellow diffusion, increased TEER values and positive ZO-1/F-actin staining post treatment, indicating improved barrier function. It was
Mo1682 Characteristics of DNA Mismatch Repair Status and Tumor Distribution Patterns in Early-Onset Colorectal Cancer Patients Juliana F. Yang, Amy Noffsinger, Jayaprakash Sreenarasimhaiah, Qinghua Yang Background/Aim: Early-onset colorectal cancers (CRCs) often occur in patients with underlying colonic disease or hereditary/genetic abnormalities such as inflammatory bowel disease (IBD) or familial CRC (Lynch syndrome, LS). Immunohistochemical detection of DNA mismatch repair (MMR) proteins is a preliminary screening tool for LS, and may play a role in determining patient prognosis and treatment. Little is known about the relationship of MMR status and CRC anatomic locations in this group of patients. Methods: The Miraca Life Sciences (MLS) pathology database was queried for all primary CRCs from 1/1/2012 to 12/31/2014. Early-onset CRC patients were those £49 years of age. The control group
S-749
AGA Abstracts
AGA Abstracts
differences in other demographics. At the time of diagnosis men had higher frequencies of cirrhosis (8.7%, 4.0%, p<0.01), previous solid organ transplant (9.0%, 4.9%, p<0.05), PEG in place (10.9%, 6.6%, p<0.05), nasogastric or orogastric tube in place (29.4%, 17.8%, p<0.001), endotracheal intubation (16.5%, 8.4%, p<0.01), and active tube feeds (21.4%, 13.1%, p<0.01) compared with woman. Men received penicillin based antibiotics (49.2%, 40.1%, p<0.05) more frequently in the three months prior to diagnosis than women with no other differences in other medication exposures. Woman had a higher WBC at diagnosis (11.6±7.7, 12.9±8.7, p<0.05) but a lower Cr (1.7±1.7, 1.5±1.6, p<0.05). There were no significant differences in albumin at diagnosis. Men received longer durations of oral metronidazole than women (11.5±9.1, 10.1±6.0 days, p<0.01) but woman had a higher frequency of switching from vancomycin to metronidazole (0.0%, 1.5%, p<0.05). Woman were discharged to home (37.6%, 39.8%, p<0.01) and readmitted within 30-days with diarrhea (18.6%, 28.7%, p<0.05) more frequently than men. There were no other significant differences in short-term outcomes, including recurrence within 90-days. Men showed a trend toward a higher 30-day mortality rate (13.4%, 11.0%, p=0.29) compared with women. Men had a higher mortality rate after one year (34.3%, 25.8%, p<0.01). Conclusions: Men get CDI less frequently, but those who do get CDI, have higher co-morbidity. Treatment patterns are similar but men have a trend toward increased short-term mortality and a higher mortality rate at one year. This might be attributed to the increased medical co-morbidities of these patients and in men, CDI should be considered an indicator of worsened potential longterm outcomes.